KR20050055717A - Methods and compositions for detection and analysis of polynucleotides using light harvesting multichromophores - Google Patents

Abstract

Methods, compositions and articles of manufacture for assaying a sample for a target polynucleotide are provided. A sample suspected of containing the target polynucleotide is contacted with a polycationic multichromophore and a sensor polynucleotide complementary to the target polynucleotide. The sensor polynucleotide comprises a signaling chromophore to receive energy from the excited multichromophore and increase emission in the presence of the target polynucleotide. The methods can be used in multiplex form. Kits comprising reagents for performing such methods are also provided.

Description

METHODS AND COMPOSITIONS FOR DETECTION AND ANALYSIS OF POLYNUCLEOTIDES USING LIGHT HARVESTING MULTICHROMOPHORES

The present invention relates to methods, articles and compositions for detecting and analyzing polynucleotides in a sample.

Methods of detecting DNA sequences in real time with high sensitivity have attracted scientific and economic interest. ^1,2,3 Their applications include medical diagnosis, identification of gene mutations, gene transfer monitoring and specific genomic techniques. Cationic organic dyes, such as ⁴ ethidium bromide and thiazole orange, emit when inserted into grooves of double stranded DNA (dsDNA) and serve as direct DNA hybridization probes, but with sequence specificity none. Energy / electron transfer chromophore pairs exist for ^5,6 strand specificity analysis, but require chemical labeling of two nucleic acids or double modification of the same modified strand (eg, molecular beacons). ^7,8 Difficult to label two DNA sites, resulting in low yield, high cost, and single labeling of impurities that lower detection sensitivity. ⁹

There is a need in the art for methods of detecting and analyzing specific polynucleotides in a sample and compositions and products useful for the methods.

Summary of the Invention

Methods, compositions, and products are provided for detecting and analyzing target polynucleotides in a sample.

Samples suspected of containing a target polynucleotide are contacted with a polycation chromophore and a sensor polynucleotide complementary to the target polynucleotide. Sensor polynucleotides include anionic backbones, such as the typical sugar phosphate backbone, and conjugate to signaling chromophores. In the presence of the target polynucleotide in the sample, the signaling chromophore can more efficiently acquire energy from the excitation polycation chromophore and emit an increased amount of detectable light or signal. Target polynucleotides may be analyzed when generated in a sample, or may be amplified prior to or in combination with analysis.

While anionic sensors can combine with cationic chromophores in the absence of a target, the double stranded complex associated with the signal generated from a mixture of non-complementary sequences due to a significant increase in signal upon binding of the target polynucleotide. It was found that The signal increase can be used for use in a detection method to analyze for a target polynucleotide in a sample.

Solutions comprising reagents usable for carrying out the methods of the invention are provided as a kit containing the reagents. The present invention also provides a signaling or complex comprising a sensing or detection complex formed from a chromophore and a sensor polynucleotide, and a target polynucleotide. The methods can be used in multiplex settings where a plurality of different sensor polynucleotides are used to analyze a plurality of different target polynucleotides. The methods can optionally be performed on a surface using surface-combined polycation chromophores; The surface may be a sensor. The methods may also be provided in a homogeneous format. The methods and articles described herein can be used as an alternative to other techniques for detecting polynucleotides.

FIG. 1 shows the absorption [a (dashed line) and c (crash)] and emission [b (square) and d (circle)] spectra of polymer (1) and fluorescein-conjugated sensor polynucleotides. . Excitation was performed at 380 nm and 480 nm for polymer (1) and sensor polynucleotides, respectively. The energy transfer complex of polymer (1) and sensor polynucleotide excited at 380 nm is shown in black (e, solid line).

FIG. 2 shows emission spectra of sensor systems containing hybridized (solid) and non-hybridized (dashed) sensor polynucleotides in 100 mmol sodium chloride and 10 mmol sodium citrate buffer at pH = 8. The spectra are normalized to the emission of the polymer 1.

FIG. 3 shows hybridization (solid line) and non-obtained from energy transfer to polynucleotide specific dyes (ethidium bromide) after excitation and energy transfer to polynucleotides [polymer (1)] to sensor polynucleotides. Emission spectra of sensor systems containing hybridized (dashed) sensor polynucleotides are shown. Measure in potassium phosphate-sodium hydroxide buffer solution (50 mM, pH = 7.40). See Example 4.

4 shows amplified emission spectra of polynucleotide-specific dyes (ethidium bromide, EB) in sensor systems containing hybridized (solid line) sensor polynucleotides. The amplified emission signal is the result of energy transfer to EB after excitation of the chromophore [polymer (1)] and energy transfer to the sensor polynucleotide. Direct EB release (dashed lines) in double-stranded DNA, and release obtained from energy transfer to EB after sensor polynucleotide excitation is shown to demonstrate the increased signal provided by polycation chromophores. Measure in potassium phosphate-sodium hydroxide buffer solution (50 mM, pH = 7.40). See Example 5.

FIG. 5 shows excitation of hybridized (solid line) and non-hybridized (dotted line) sensor polynucleotides in polymer (1) and potassium phosphate-sodium hydroxide buffer solution (50 mM, pH = 7.40) in the presence of EB. The emission spectrum of EB is shown. See Example 6. Release is the result of energy transfer from the polycation chromophore to the inserted EB only in the case of hybridized double stranded DNA.

Current techniques for DNA and RNA sensors (including "gene-chips" and "DNA-chips") are based on fluorescent tags for single strands of DNA, depending on the label of the sample or sample component. There was an inevitable problem that the efficacy of the marker response of the sample-sample, which depends on the covalent bond of the lumophore], requires complex cross-correction. Other systems augment labeled probes that need to multiplex attach lumopores and quenchers to correctly engineered sequences (eg, molecular beacons, Taqman, Trademark Scorpion). , Trade name), and probes Eclipse probe.

The method of the present invention comprises contacting a sample with an aqueous solution comprising two or more of the following components:

(a) condensing, polycationic, luminescent chromophore systems such as conjugated polymers, water soluble semiconductor quantum dots or dendritic structures; And (b) sensor polynucleotides (also referred to as "oligo-C ^* ") conjugated to luminescent signaling chromophores.

The emission of light with wavelength characteristics of the signaling chromophore indicates that it is present in a solution of the target polynucleotide having a base sequence complementary to that of the sensor polynucleotide. By using a plurality of sensor polynucleotides each having a different base sequence and different signaling chromophores (oligo ₁ -C ₁ ^* , oligo ₂ -C ₂ ^*, etc.), a plurality of polynucleotides each having specific base sequences can be independently Can be detected. A third component, such as a polynucleotide-specific dye, can be introduced to improve selectivity by further transferring energy from the sensor polynucleotide to the polynucleotide-specific dye.

The condensing chromophore and the signaling chromophore C ^* are selected such that the absorption bands of the two species overlap to a minimum, and the luminescent emission spectra of the two species are present at different wavelengths. When prepared in aqueous solution, condensing and luminescent chromophore systems are positively charged (eg positively charged conjugated polyelectrolytes). Proximity between the negatively charged sensor polynucleotide and the positively charged condensing and luminescent chromophore system is ensured by electrostatic attraction. When exposed to incident radiation having a wavelength in the absorption band of the condensed chromophore, it is emitted from the signal chromophore via a Fosterster energy transfer mechanism. When adding a target polynucleotide, such as single-stranded DNA (ssDNA) having a complementary base sequence to the sequence of the sensor polynucleotide, the ssDNA hybridizes with the sensor polynucleotide to increase the negative charge density along the DNA strand. ^{10,11 Under the} above circumstances, and taking into account electrostatic forces, the interaction between the sensor polynucleotide and the positively charged condensed chromophore system would be desirable, thereby enabling efficient energy transfer and intense emission from the signaling chromophores. Base pair hybridization does not occur when ssDNA is added with a base sequence that is not complementary to the sequence of the sensor polynucleotide. Electrostatic complexation between the condensed chromophore system and the sensor polynucleotide is competitively screened with non-complementary ssDNA. Thus, the average distance between the condensed chromophore system and the oligo-C ^* becomes large in the presence of non-complementary sequences, thus making energy transfer to the signaling chromophores less effective. The release of signaling oligo-C ^* is greater in the presence of its complementary target than in the presence of a non-complementary strand. The overall scheme provides a sensor for the presence of the target polynucleotide with a specific base sequence in the test solution. By using a plurality of sensor polynucleotides each having a different nucleotide sequence and a different signaling chromophore, the plurality of targets can be detected separately.

In addition to the above method, the present invention provides an aqueous solution comprising two or more of the following components:

(a) cationic chromophores; And (b) “sensor polynucleotides” (oligo-C ^* ) comprising anionic polynucleotides conjugated to a signaling chromophore.

As demonstrated in the examples, optical amplification provided by water soluble chromophores, such as conjugated polymers, can be used to detect polynucleotide hybridization to sensor polynucleotides. Amplification can be improved using high molecular weight water soluble conjugated polymers or structures other than the polycationic polychromophores described herein. The present invention can be provided in a uniform format that facilitates the use of fluorescence detection. The present invention can be used to detect amplified target polynucleotides as a stand alone assay without the need to amplify the polynucleotides because of the large signal amplification.

Unlike gene-chip technology, the present invention does not necessarily require a marker for each sample to be analyzed by covalently binding a lumophore or chromophore to polynucleotides contained in or derived from the sample prior to analysis. The binding methods inherently have difficulty in reproducing binding efficacy and require sample-sample cross-correction.

The present invention can be used in any assay as long as the sample can be queried with respect to the target polynucleotide. Typical assays include methods for determining the presence or relative amounts of target polynucleotides in a sample, or can be quantitative or semi-quantitative.

The methods of the present invention can be performed in a multiplex format. A plurality of different sensor polynucleotides can be used to detect corresponding other target polynucleotides in a sample by using different signaling chromophores conjugated to each sensor polynucleotide. 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 200, 400 that can be used simultaneously to analyze for corresponding other target polynucleotides Multiplex methods are provided that use two or more different sensor polynucleotides.

The methods can be carried out in solution as well as in substrate, but the solution form is expected to be faster due to diffusion problems. Thus, the assay can be performed in the form of an array on a substrate, which can be a sensor. This can be accomplished by immobilizing or incorporating the assay component onto a substrate, such as a sensor polynucleotide, a polycation chromophore, or both. The substrates may be surfaces of glass, silicon, paper, plastic, or optoelectronic semiconductors (such as, but not limited to, indium-doped gallium nitride or polymerizable polyaniline) used as optoelectronic transducers. Can be. The position of a given sensor polynucleotide is known or measurable in the form of an array, which is microaddressable or nanoaddressable. In one variation, one or more samples containing amplification products can be attached to a substrate, and the substrate can be contacted with one or more labeled sensor polynucleotides and polycationic polychromophores.

Before describing the invention in more detail, it is understood that the invention is not limited to any particular method, apparatus, solution or apparatus, as these methods, apparatus, solutions or apparatus may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not to be regarded as limiting the scope of the invention.

Using the singular forms includes the plural unless the context clearly dictates otherwise. Thus, "target polynucleotide" includes a plurality of target polynucleotides, "signaling chromophore" includes a plurality of said chromophores, and "sensor polynucleotide" includes a plurality of sensor polynucleotides. In addition, the use of specific plural forms such as "two", "three", and the like is read to many identical objects unless the context clearly dictates.

Terms such as "connected", "attached" and "coupled" are used interchangeably herein and include direct as well as indirect linking, attachment, binding or conjugation unless the context clearly dictates. do. When a range of values is cited, each intervening integer value, and each fraction thereof between the upper and lower limits of the range, is specified along each subrange between the values. The upper and lower limits of the range may be included or excluded independently within the range, and each range including either limit value, any limit value or all limit values is included in the present invention. If the value has an inherent limit, for example if the component is present at a concentration of 0% to 100% or if the pH of the aqueous solution can be in the range of 1 to 14, the inherent ranges are specified. Where the values are expressly defined, it is understood that values relating to the same quantity or quantity as the recited values are within the scope of the present invention and ranges based thereon. Where combination examples are described, subcombinations of the combination elements are described specifically and are within the scope of the present invention. Conversely, in the case where other elements or element groups are disclosed, combinations thereof are also disclosed. When any element of the invention is described as having a plurality of alternatives, embodiments of the invention are also disclosed in which each alternative is excluded alone or in combination with other alternatives; One or more elements of the invention may have the above exclusions, and all element combinations having the above exclusions are also disclosed.

Unless otherwise specified or clearly stated in the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

All documents mentioned herein are hereby incorporated by reference for the purpose of disclosing and describing the specific materials and methods for which reference is cited. Publications disclosed herein are those disclosed prior to the filing date of the present application. It is not considered that the present invention is deemed to have no right to hasten the foregoing disclosure by conventional invention.

Definitions

In describing the present invention, the following terms will be used and are intended to be defined as described below.

Terms such as "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule" are intended to mean a polymerized form of nucleotides of a certain length. Possible use may include ribonucleotides, deoxyribonucleotides, analogs thereof or mixtures thereof. The terms refer only to the primary structure of the molecule. Thus, triple stranded, double stranded and single stranded ribonucleic acid (“RNA”) as well as triple stranded, double stranded and single stranded deoxyribonucleic acid (“DNA”) are included. Also included are polynucleotides in modified and unmodified form by alkylation and / or capping.

If modified or unmodified, the sensor polynucleotide is anionic and can interact with the cationic polychromophores in the absence of the target polynucleotide. The target polynucleotide may or may not be charged, but is typically expected to be anionic RNA or DNA.

In particular, terms such as "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule" contain (2-deoxy-D-ribose) Polydeoxyribonucleotides, polynucleotides (containing D-ribose), spliced or unspliced tRNAs, rRNAs, hRNAs and mRNAs, and purine bases or pyrimidines Other kinds of polynucleotides that are N-glycosides or C-glycosides of bases, and other polymers containing phosphate or other polyanion backbones, and other synthetic sequence-specific nucleic acid polymers, provided that the polymers are DNA and It contains nucleobases in an array for base pairing and base stacking as found in RNA. Terms such as "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule" are not distinguishable in length, and the terms are used interchangeably herein. The terms refer only to the primary structure of the molecule. Thus, the terms are for example 3'-deoxy-2 ', 5'-DNA, oligodeoxyribonucleotide N3'P5' phosphoramidate, 2'-0-alkyl-substituted RNA, double Strands and single-stranded DNA, as well as double-stranded and single-stranded RNA, and hybrids thereof, including hybrids between DNA and RNA, and also known forms of modifications, for example Label, alkylation, “cap”, substitution, one or more nucleotide modifications of one or more nucleotides by an analog, eg, a modification with negatively charged bonds (eg, phosphoro) Thioate, phosphorodithioate, etc.), and modifications containing pendant components, such as, for example, [enzymes (eg, nucleases), toxins, antibodies, signal peptides, poly-L-lysine, etc.). Protein], modified with an intercalator (eg, acridine, soraren len), etc.), chelate-containing variants (e.g., metals, radioactive metals, boron, oxidative metals, etc.), alkylating agent-containing variants, variants with modified bonds (e.g., α anomer nucleic acid And the like, as well as polynucleotides or oligonucleotides in unmodified form.

As used herein, terms such as "nucleoside" and "nucleotide" refer to components that include known purine bases and pyrimidine bases, as well as modified other heterocyclic bases. It is understood to include. Such modified bases include methylated purines or pyrimidines, acrylated purines or pyrimidines, or other heterocyclics. Modified nucleosides or nucleotides also include modifications in the sugar component, wherein one or more hydroxyl groups are substituted with halogen, aliphatic groups, and can be functionalized by ethers, amines, and the like. "Nucleotidic units" tend to include nucleosides and nucleotides.

Modifications to nucleotide units also include rearrangements, additions, substitutions to alter functional groups on the purine base or pyrimidine base that form hydrogen bonds to the complementary pyrimidine or purine, respectively. The resulting modified nucleotide units can optionally form base pairs with said other modified nucleotide units, but cannot form base pairs with A, T, C, G or U. Abasic sites can be incorporated so as not to interfere with the function of the polynucleotides; Preferably the polynucleotide does not comprise a nonbase position. Some or all of the residues in the polynucleotides may optionally be modified in one or more ways.

Standard AT and GC base pairs are between N3-H and C4-oxy of thymidine and N1 and C6-NH2 of adenosine respectively, and C2-oxy, N3 and C4-NH2 of cytidine, and C2-NH2, N 'of guanosine It is formed under conditions such that a hydrogen bond is formed between -H and C6-oxy. Thus, for example, guanosine (2-amino-6-oxy-9-β-D-ribofuranosyl-purine) is modified so that isoguanosine (2-oxy-6-amino-9-β-D-ribofura Nosyl-purine) can be formed. This modification will no longer form standard base pairs with cytosine at the nucleoside base. However, modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) results in isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine). ) Will no longer effectively form base pairs with guanosine in the modified nucleotides but will form base pairs with isoguanosine. Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); Isocytidine can be prepared by the methods described in Switzer et al. (1993) Biochemistry 32: 10489-10496 and references cited therein; 2'-deoxy-5-methyl-isocytidine is described by Tor et al. (1993) J. Am. Chem. Soc. 115: 4461-4467 and the references cited therein; And isoguanine nucleotides described above by Switzer et al. (1993) and by Mantsch et al. (1993) Biochem. 14: 5593-5601 or US Pat. No. 5,780,610 to Collins et al. Other unnatural base pairs are described in Piccirilli et al. (1990) Nature 343: 33-37 with 2,6-diaminopyrimidine and its complement (1-methylpyrazolo- [4,3] pyrimidine-5,7- ( 4H, 6H) -dione) can be synthesized by the method described for synthesis. Other modified nucleotide units that form unique base pairs are known and are described in Leach et al. (1992) J. Am. Chem. Soc. 114: 3675-3683 and Switzer et al., Supra.

"Preferential binding" or "preferential hybridization" indicates an increase in the tendency of one polynucleotide or PNA in a sample to bind its complement to a non-complementary polymer in the sample.

Hybridization conditions typically include salts at concentrations of about 1 M or less, more typically about 500 mM or less, preferably about 200 mM or less. In the case of hybridization between the peptide nucleic acid and the polynucleotide, the hybridization can be carried out in a solution containing little or no salt. The hybridization temperature is 5 ° C. or less, but is typically at least 22 ° C., more typically at least about 30 ° C., and preferably at least about 37 ° C. Longer fragments may require higher hybridization temperatures in specific hybridizations. Other factors can affect hybridization requirements, including, for example, base composition and length of complementary strands, presence of organic solvents, and degree of base mismatch, and the combination form of the parameters used may be More important than absolute measurement. Suitable hybridization conditions for a given assay form can be determined by one of ordinary skill in the art: non-limiting parameters that can be controlled include analyte components, salts used and concentrations of these concentrates, ionic strength, Presence and concentration of a blocking agent that reduces background binding, such as temperature, buffer form and concentration, solution pH, repeat sequence, or presence and concentration of protein solution blocker, detergent form (s) and concentration, polynucleotide Molecules such as polymers that increase relative concentrations, metal ion (s) and their concentrate (s), chelating agent (s) and their concentrates and other conditions known in the art.

"Multiplexing" herein refers to an analytical method that can analyze multiple analytes simultaneously.

The expressions "optional" or "optionally" are understood to be cases or situations that may or may not occur in the following description, and examples and examples do not occur in the specification. Examples do not include.

Sample

The portion of the sample that contains or is believed to contain the target polynucleotide is directly from the organism (including cells, tissues or fluids) and the deposits left by the organism (including viruses, mycoplasmas and fossils). Or a biological material comprising a polynucleotide that can be obtained indirectly can be a source. The sample may comprise some or all of the target polynucleotide prepared via synthetic means. Typically, the sample can be obtained mainly in the form of an aqueous medium or in a form dispersed in an aqueous medium. Non-limiting examples of the sample include blood, urine, semen, milk, sputum, mucus, buccal swab, vaginal swab, rectal swab, aspirate ( aspirate, needle biopsy, tissues obtained by surgery or autopsy, plasma, serum, spinal fluid, lymph, skin, respiratory organs, external secretions of the intestine and urogenital tract, tears, saliva, tumors, organs, tests Samples of in vitro cell culture components (including but not limited to cell culture media, conditioned media resulting from the growth of cells in putative viral infected cells, recombinant cells and cellular components), and polynucleotide sequences Recombinant library is included. Samples may be present on the substrate as described herein. The substrate can be a slide containing the sample and used for fluorescence in situ hybridization (FISH).

The sample can be a positive control sample known to contain the target polynucleotide or surrogate thereof. Although it is not expected to include the target polynucleotide, it is thought to include it (through contamination of one or more reagents) or other components that can produce artificial positives and target the reagents used in a given assay. Negative control sample tested to determine the lack of contamination by polynucleotides as well as to determine if a given set of assay conditions produce artificial positives (positive signals in the absence of target polynucleotides in the sample). ) Can also be used.

The sample can be diluted, dissolved, suspended, extracted, or otherwise processed to dissolve and / or purify the target polynucleotide present, or make accessible to the reagents or detection reagents used for amplification. The sample comprises cells, which cells can be degraded or infiltrated to release polynucleotides within the cells. One step permeabilization buffer is used to degrade the cells and an additional step, for example a polymerase chain reaction, is performed immediately after the degradation.

Target polynucleotides and amplification products generated therefrom

The target polynucleotide can be single stranded, double stranded, or more stranded, and can be straight or cyclic. Examples of single stranded target polynucleotides include mRNA, rRNA, tRNA, hnRNA, ssRNA or ssDNA viral genomes, which may contain complementary sequences and important secondary structures therein. Examples of double stranded target polynucleotides include genomic DNA, mitochondrial DNA, chloroplast DNA, dsRNA or dsDNA viral genomes, plasmids, phages and viroids. The target polynucleotide can be prepared synthetically or can be purified from a biological source. The target polynucleotide can be purified to reduce or eliminate one or more unwanted components of the sample or to concentrate the target polynucleotide. Conversely, if the target polynucleotide is too concentrated for a particular assay, the target polynucleotide can be diluted.

Following sample collection and selective nucleic acid extraction, the nucleic acid portion of the sample comprising the target polynucleotide may be treated with one or more preliminary reactions. Preliminary reactions include in vitro transcription (IVT), labeling, fragmentation, amplification and other reactions. mRNA is prepared by treatment with reverse transcriptase and primers first prior to detection and / or amplification to prepare cDNA: in situ, such as in cells or tissues that have been run with purified mRNA in vitro or immobilized on slides. ). Nucleic acid amplification increases the copy number of the desired sequence, such as the polynucleotide of the target. Various amplification methods are suitable for use; Non-limiting examples of suitable amplification reactions include polymerase chain reaction (PCR), ligase chain reaction (LCR), self sustained sequence replication (3SR), nucleic acid sequence amplification (NASBA), Use of Q beta transcriptase, reverse transcription, nick translation, and the like.

If the target polynucleotide is single stranded, the first amplification cycle forms a primer extension product complementary to the target polynucleotide. If the target polynucleotide is a single stranded RNA, a polymerase with reverse transcriptase activity can be used for the first amplification to reverse transcribing from RNA to DNA and an additional amplification cycle can be performed to copy the primer extension product. Primers for PCR will be designed to hybridize to their corresponding intratemplate regions to form amplifiable segments; Thus each primer is hybridized so that its 3 'nucleotide is paired with a nucleotide in its complementary template strand and is located 3' from the 3 'nucleotide of the primer used to replicate the complementary template strand during PCR.

Target polynucleotides are typically amplified by contacting one or more strands of the target polynucleotide with polymerases and primers with suitable activity to extend the primers, and copy the target polynucleotides to complete a full length complementary polynucleotide or portion thereof. Create Enzymes having polymerase activity capable of copying target polynucleotides can be used, including, for example, DNA polymerase, RNA polymerase, reverse transcriptase, enzymes having at least one polymerase activity, the enzyme being thermolabile (thermolabile) or thermostable (thermostable). Mixtures of enzymes may also be used. Examples of enzymes include DNA polymerases (eg, DNA polymerase I ("Pol I"), Klenow fragments of Pol I, T4, T7, Sequenase® T7, Squanase®) version 2.0 T7, Tub, Taq, Tth , Pfx, Pfu, Tsp, Tfl, Rhodococcus species (Pyrococcus sp) GB-D DNA polymerase] by Tli and pastries; RNA polymerases (eg, E. coli , SP6, T3, and T7 RNA polymerases); And reverse transcriptases (eg, AMV, M-MuLV, MMLV, RNAse H ^- MMLV (SuperScript®), SuperScript® II, ThermoScript®, HIV-1, and RAV2 reverse transcriptase). All the said enzyme can use a commercial item. Examples of polymerases having multiple specificities include RAV2 and Tli (exo-) polymerases. Examples of thermostable polymerases include Lactococcus species GB-D DNA polymerase as Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tli and pies.

Suitable reaction conditions include pH, buffer, ionic strength, the presence and concentration of one or more salts, the presence and concentration of reactants and cofactors (such as nucleotides and magnesium and / or other metal ions (eg manganese)), optional cosolvents ( Amplification of the target polynucleotide by selecting a thermal cycling profile for amplification, including cosolvent, temperature, polymerase chain reaction, and may depend on the nature of the sample as well as on where the polymerase is used. Cosolvents include formamide (typically from about 2% to about 10%), glycerol (typically from about 5% to about 10%), and DMSO (typically from about 0.9% to about 10%). Amplification techniques can be used to minimize the production of artifacts generated during artificial positive or amplification. These include "touchdown" PCR, hot-start techniques, use of nested primers or designed PCR primers, which are stem-loops in the case of primer-dimer formation It does not amplify by forming a (stem-loop) structure. As a technique for promoting PCR, centrifugal PCR (which causes greater convection in the sample) can be used and includes an infrared heating step for rapid heating and cooling of the sample. One or more amplification cycles may be performed. One or more primers are used to generate one or more primer extension products during PCR; Preferably the primer extension product produced in excess is the amplification product detected. Several different primers can be used to amplify other target polynucleotides or other regions of a particular target polynucleotide in the sample.

The amplified target polynucleotide may be processed after the amplification treatment. For example, in some cases it is desirable to fragment the target polynucleotide prior to hybridization to provide a more easily accessible segment. Fragmentation of the nucleic acid can be carried out by a method of producing fragments of a size useful in the assays performed; Suitable physical, chemical and enzymatic methods are known in the art.

The amplification reaction can be carried out under conditions that hybridize the sensor polynucleotides to be an amplification product during some or all of the amplification cycle. The analysis can be carried out by the method and by monitoring the change in light emission from the signal chromophore generated by the hybridization during amplification with real time detection of the hybridization.

Polycationic multichromophore

Condensed chromophore systems have proven to be effective light absorbers containing multiple chromophores. Examples thereof include, but are not limited to, conjugated polymers, aggregates of conjugated molecules, luminescent dyes attached to saturated polymers, semiconductor quantum dots, and dendritic structures. For example, on a conjugated polymer, each repeating unit can be considered as a distributed chromophore, a quantum dot consists of many atoms, a saturated polymer can be functionalized by many luminescent dye molecules in the side chain, and a dendrimer ) Can be synthesized to include individual chromophores with many covalent bonds. It can be used for condensing by attaching a chromophore assembly onto a solid support (such as a polymer bead or surface).

A condensed chromophore system can effectively transfer energy to the next luminescent species (eg, "signaling chromophores"). Energy transfer mechanisms include, for example, resonance energy transfer (Foerster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), and the like. Typically, however, the energy transfer mechanism is relatively short in scope; That is, the approach of the condensed chromophore system to the signal chromophore is required for efficient energy transfer. Under conditions for efficient energy transfer, amplification of the emission from the signaling chromophore occurs when the number of individual chromophores in the condensed chromophore system increases; That is, the emission from the signal chromophore is stronger when the incident light (“pump light”) is at a wavelength absorbed by the condensed chromophore system than the signal chromophore is directly excited by the pump light.

Conjugated polymers (CPs) feature unlocalized electronic structures and can be used as highly responsive optical reporters for chemical and biological targets. ^{12,13 Since the} efficient conjugate length is substantially shorter than the polymer chain, the backbone comprises a large amount of conjugated segments. Therefore, the conjugated polymer is efficient for condensing and can amplify light through Foster energy transfer. ¹⁴

Spontaneous interpolymer complexation between cationic polyelectrolyte and DNA is disclosed, which produces a common electrostatic force. Hydrophobic reactions between ^15,16,17 aromatic polymer units and DNA bases have also been recently known. The free energy of the ^18,19 polyelectrolyte / DNA reaction is controlled by the structure of the participating molecular species used in combination with the solution variables (eg pH, ionic strength and temperature). ²⁰ The intensity and specificity of the reaction was recently adjusted to recognize the tertiary structure of plasmid DNA. ²¹

The chromophores used in the present invention are polycations so that they can react electrostatically with sensor polynucleotides. Polycation chromophores that can absorb light and can transfer energy to signal-indicating chromophores on sensor polynucleotides can be used in the described methods. Examples of chromophores that can be used include conjugated polymers, saturated polymers or dendrimers, and semiconductor nanocrystals (SCNCs) comprising multiple chromophores in various ways. Conjugated polymers, saturated polymers, and dendrimers can be prepared and incorporated into or derivatized with a plurality of cationic molecular species to make them polyionic after synthesis; Semiconductor nanocrystals can be made into polycations by adding cationic molecular species to their surfaces.

In a preferred embodiment, the conjugated polymer is used as a polycationic polychromophore. Specific examples are disclosed in Formula 1 wherein the cationic water soluble conjugated polymer is a poly ((9,9-bis (6'-N, N, N-trimethylammonium) -hexyl) -fluorene with iodide covalent Phenylene) (denoted as polymer (1) below). ²² The specific size of the polymer is not critical, but it must be able to absorb light and transfer energy to bring the chromophore closer to the signal. Typical values of "n" are 2 to about 100,000. The specific molecular structure is not important; Water soluble cationic conjugated polymers with sufficient luminescence quantum efficiency are used.

Water soluble conjugated oligomers can also be used as polycation chromophores. Examples of the water-soluble cation-emitting conjugated oligomers having iodide counterions are disclosed in Formula 2 below (represented by oligomer (2)).

Although smaller oligomers 2 do not exhibit the large signal amplification properties of high molecular weight polymers, small molecules are useful for deconvolute structural property relationships, and inherent polydispersity and placement found in polymers. It is difficult to determine batch-to-batch variation. In addition, in aqueous media, oligomers (eg, formula (2)) are more soluble than their polymeric counterparts, and hydrophobic reactions in the oligomer (2) are expected to be of less importance in the polymer structure. It may therefore be desirable to use assemblies of oligomers in certain applications. Organic solvents (eg, water soluble organic solvents such as ethanol) may be added to reduce background C ^* release. The presence of organic solvents can reduce hydrophobic reactions and reduce background C ^* release.

Sensor polynucleotide

Sensor polynucleotides are anionic, provide complementarity to target polynucleotides to be analyzed and have a selected sequence. The sensor polynucleotides may be branched, multimeric, or cyclic, but are typically straight chain and may include nonnatural bases. Sensor polynucleotides can be prepared with the desired base sequence. Chemical methods for attaching signaling chromophores to sensor polynucleotides are well known. Specific sensor polynucleotide structures, including structures conjugated to ²³ chromophores, can be prepared using commercially available or chemically synthesized ones.

Signaling chromophore

Chromophores useful in the present invention described above include materials capable of absorbing energy and emitting light from polycation chromophores in a suitable solution. For multiplexing analysis, several different signaling chromophores can be used with different detectable emission spectra. Chromophores can be lumophores or fluorophores. Typical fluorophores include fluorescent dyes, semiconductor nanocrystals, lanthanide chelates, polynucleotide-specific dyes and green fluorescent proteins.

Examples of fluorescent dyes include fluorescein, 6-FAM, rhodamine, Texas red, tetramethyltamine, carboxyrodamine, carboxyrodamine 6G, carboxylodol, carboxyrodamine 110, cascade Cascade Blue, Cascade Yellow, Komarin, Cy2 (R), Cy3 (R), Cy3.5 (R), Cy5 (R), Cy5.5 (R), Cy-Chrome, Pico Erythrin, PerCP (ferridinine chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), NED, ROX ( 5- (and-6) -carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green (Oregon Green) 514, Alexa Fluor (trade name) 350, Alexa Floor (trade name) 430, Alexa Floor (trade name) 488, Alexa floor (trade name) 532, Alexa floor (trade name) 546, Alexa Floor (trade name) 568, Alexa floor (trade name) 594, Alexa floor (trade name) 633, Alexa floor (trade name) 647, Alexa floor (trade name) 660, Alexa floor (trade name) 680, 7-amino-4-methylcomarin -3-acetic acid, Bodypie (BODIPY, brand name) FL, Bodypie (trade name) FL-Br ₂ , Bodypie (trade name) 530/550, Bodypie (trade name) 558/568, Bodypie (trade name) 564/570 , Bodypie (trade name) 576/589, Bodypie (trade name) 581/591, Bodypie (trade name) 630/650, Bodypie (trade name) 650/665, Bodypie (trade name) R6G, Bodypie (trade name) TMR , Bodypie TR, conjugated products thereof, and combinations thereof. Examples of lanthanide chelates include europium chelates, terbium chelates, and samarium chelates.

Various fluorescent semiconductor nanocrystals (SCNCs) are known in the art; Methods for making and using semiconductor nanocrystals are disclosed in PCT Publication No. WO99 / 26299 (published May 27, 1999) by Bawendi et al., USPN 5,990,479 (registered November 23, 1999) by Weiss et al. And Bruchez et al. Science 281: 2013 (1998). Semiconductor nanocrystals are obtained with very narrow emission bands with well-defined peak emission wavelengths, allowing many different SCNCs to be used as signal chromophores in the same assay, optionally combining with other non-SCNC type signaling chromophores. .

Typical polynucleotide-specific dyes are acridine orange, acridine homodimer, actinomycin D, 7-aminoactinomycin D (7-AAD), 9-amino-6-chloro-2 -Methoxyacridine (ACMA), BOBO (trade name) -1 iodide (462/481), BOBO (trade name) -3 iodide (570/602), BO-PRO (trade name) -1 iodide (462 / 481), BO-PRO ™ -3 Iodide (575/599), 4 ', 6-Diamidino-2-phenylindole, Dihydrochloride (DAPI), 4', 6-Diamidino-2 -Phenylindole, dihydrochloride (DAPI), 4 ', 6-diamidino-2-phenylindole, dilactate (DAPI, dilactate), dihydroethidium (hydroetitidine), dihydroethdium (hydro Etidine), dihydroethidium (hydroetitidine), ethidium bromide, ethidium diazide chloride, ethidium homodimer-1 (EthD-1), ethidium homodimer-2 (EthD-2) , Ethidium monoazide bromide (EMA), hexydium iodide, Hoechst 33258, Hoechst 333 42, Hoechst 34580, Hoechst S769121, hydroxysteelbamidine, methanesulfonate, JOJO®-1 iodide (529/545), JO-PRO®-1 iodide (530/546), LOLO ( Trademark name-1 Iodine (565/579), LO-PRO (trade name) -1 Iodine (567/580), NeuroTrace (trade name) 435/455, NeuroTrace (trade name) 500/525, NeuroTrace (trade name) 515 / 535, NeuroTrace (R) 530/615, NeuroTrace (R) 640/660, OliGreen, PicoGreen (R) ssDNA, PicoGreen (R) dsDNA, POPO (R) (1) Iodine (434/456), POPO (R) -3 Iodide (534/570), PO-PRO (trade name) -1 Iodide (435/455), PO-PRO (trade name) -3 Iodide (539/567), propidium iodide, RiboGreen (trade name) ), SlowFade (trade name), SlowFade (trade name) Light , SYBR (trade name) Green I, SYBR (trade name) Green II, SYBR (trade name) Gold, SYBR (trade name) 101, SYBR (trade name) 102, SYBR (trade name) 103 , SYBR (trade name) DX, TO-PRO (trade name) -1, TO-PRO (trade name) -3, TO-PRO (trade name) -5, TOTO (trademark) Name) -1, TOTO (trade name) -3, YO-PRO (trade name) -1 (oxazole yellow), YO-PRO (trade name) -3, YOYO (trade name) -1, YOYO (trade name) -3, TO , SYTOX (trade name) Blue, SYTOX (trade name) Green, SYTOX (trade name) Orange, SYTO (trade name) 9, SYTO (trade name) BC, SYTO (trade name) 40, SYTO (trade name) 41, SYTO (trade name) 42, SYTO (Trade name) 43, SYTO (trade name) 44, SYTO (trade name) 45, SYTO (trade name) Blue, SYTO (trade name) 11, SYTO (trade name) 12, SYTO (trade name) 13, SYTO (trade name) 14, SYTO (trade name) ) 15, SYTO (trade name) 16, SYTO (trade name) 20, SYTO (trade name) 21, SYTO (trade name) 22, SYTO (trade name) 23, SYTO (trade name) 24, SYTO (trade name) 25, SYTO (trade name) Green , SYTO (trade name) 80, SYTO (trade name) 81, SYTO (trade name) 82, SYTO (trade name) 83, SYTO (trade name) 84, SYTO (trade name) 85, SYTO (trade name) Orange, SYTO (trade name) 17, SYTO (Trade name) 59, SYTO (trade name) 60, SYTO (trade name) 61, SYTO (trade name) 62, SYTO (trade name) 63, SYTO (trade name) 64, SYTO (trade name) Red, netropsin, distamycin, acri Include orange, 3,4-benzopyrene, thiazole orange, TOMEHE, daunomycin, acridine, pentyl -TOTAB and butyl -TOTIN. Asymmetric cyanine dyes can be used as polynucleotide-specific dyes. Other dyes of interest are Geierstanger, BH and Wemmer, DE, Annu. Rev. Vioshys. Biomol. Struct. 1995, 24, 463-493, by Larson, CJ and Verdine, GL, Bioorganic Chemistry: Nucleic Acids, Hecht, SM, Ed., Oxford University Press: New York, 1996; pp 324-346, and by Glumoff, T. and Goldman, A. Nucleic Acids in Chemistry and Biology, 2 ^nd ed., Blackburn, GM and Gait, MJ, Eds., Oxford University Press: Oxford, 1996, pp375-441 It includes those described in. The polynucleotide-specific dyes can be insertion dyes and can be specific for double-stranded polynucleotides. Other dyes and fluorophores are described at www.probes.com (Molecular Probes, Inc.).

The term "green fluorescent protein" refers to a native version of the Aequorea green fluorescent protein and a mutated version that is recognized to exhibit altered fluorescence properties, including altered excitation and maximal emission, and other forms of excitation. And emission spectra (Delagrave, S. et al. (1995) Bio / Technology 13: 151-154; Heim, R. et al. (1994) Proc, Natl. Acad. Sci. USA 91: 12501-12504 Heim, R. et al. (1995) Nature 373: 663-664). Delgrave et al. Isolated variants of cloned Aequorea victoria GFP with red-transition excitation spectra. Bio / Technology 13: 151-154 (1995), Heim R. et al. Reported variants with blue fluorescence (Tyr 66 to His) (Proc. Natl. Acad. Sci. (1994) USA 91: 12501-12504). .

 In one variation, a second signaling chromophore, which may be attached directly or indirectly to another assay component and / or substrate, is used to receive energy from the initial signaling chromophore. In certain applications, this may provide significant additional selectivity. For example, polynucleotide-specific dyes can be used as initial or second signaling chromophores and can be specific for double-stranded sequences. The energy can then be transferred from the excited cation chromophore to the initial signaling chromophore, after which the energy is transferred to the second signaling chromophore in the overall format selectable to the target. The cascade of signaling chromophores can in principle be extended to use any of a number of signaling chromophores with appropriate absorption and emission profiles. In one embodiment of this variant, the insertion dye specific for the double-stranded polynucleotide is used as a second signaling chromophore, and the initial signaling chromophore capable of transferring energy to the second signaling chromophore is a sensor polynucleotide. Is conjugated to. Insertion dyes provide an additional optional requirement to hybridize the sensor and the target polynucleotide prior to gathering in the detection complex. In the presence of the target, duplexes are formed, dyes are collected, and signaling from the second signaling chromophore is induced by excitation of the chromophore.

Substrate

The methods described herein can be carried out on various types of substrates. The substrate may comprise a broad range of materials, biological, abiotic, organic, inorganic or a combination thereof. For example, the substrate may be a variety of polymerizable Langmuir Blodgett films, functionalized glass, Si, Ge, GaAs, GaP, SiO ₂ , SiN ₄ , modified silicon, gels or polymers such as (poly) Tetrafluoroethylene, (poly) vinylidenedifluoride, polystyrene, crosslinked polystyrene, polyacrylic, polylactic acid, polyglycolic acid, poly (lactide coglycolide), polyanhydride, poly (methyl methacrylate) , Poly (ethylene-co-vinyl acetate), polysiloxane, polymerizable silica, latex, dextran polymer, epoxy, polycarbonate, agarose, poly (acrylamide) or combinations thereof. Conducting polymers and photoconductive materials can be used.

Substrates are planar crystalline materials such as silica based materials (eg glass, quartz, etc.), or crystalline materials used in the semiconductor and microprocessor industries, such as silicon, gallium arsenide, indium doped GaN, etc. This includes semiconductor nanocrystals.

The substrate may take the form of a photodiode, an optoelectronic sensor, for example in the form of an optoelectronic semiconductor chip or an optoelectronic thin film semiconductor, or a biochip. The position (s) of the individual sensor polynucleotide (s) on the substrate may be addressable; This may be done in a high density format and the location may be microaddressable or nanoaddressable.

Silica aerogels can also be used as substrates and prepared by methods known in the art. Airgel substrates can be used as surface coatings for free standing substrates or other substrate materials.

Substrates can be in any form and are typically flat, slide, bead, pellet, disk, particle, microparticle, nanoparticle, strand, precipitate, optionally porous gel, sheet, tubular , Sphere, container, capillary, pad, slice, film, chip, multiwell plate or dish, optical fiber, and the like. The substrate may be in a solid or semi-solid form. The substrate may comprise raised or lowered regions in which sensor polynucleotides or other analytical components are located. The surface of the substrate is corroded by known techniques to provide the desired surface properties (eg trenches, V-grooves, mesa structures, etc.).

The substrate surface may be made of the same material as the substrate or may be made of another material and may be bonded to the substrate by chemical or physical means. The bonded surface may be made of a variety of materials, for example polymers, plastics, resins, polysaccharides, silica or silica based materials, carbon, metals, inorganic glasses, membranes, or any of the substrate materials. have. The surface may optionally be transparent and may have surface Si—OH functionality such as that found on the silica surface.

The substrate and / or its optional surface is selected to provide suitable optical properties in the synthesis and / or detection method used. The substrate and / or surface may be transparent to expose the substrate by light applied from multiple directions. The substrate and / or surface may have a reflective “mirror” structure to increase the recovery of light.

The substrate and / or its surface is generally resistant to, and treated to, the conditions that should be exposed during use, and may optionally be treated to remove any resistant material after exposure to the conditions.

Sensor polynucleotides may be prepared by any suitable method, for example US Pat. No. 5,143,854, PCT Publication No. WO 92/10092, US patent application Ser. No. 07 / 624,120 (filed Dec. 6, 1990, now abandoned), Fodor et al., Science, 251: 767-777 (1991), and PCT Publication No. It can be attached to or prepared on a substrate by the method described in WO 90/15070. Techniques for synthesizing the arrays using a mechanical synthesis method are described, for example, in PCT Publication No. WO 93/09668 and US patent no. 5,384,261.

Further techniques are also described in PCT Application No. Beads based on the same techniques as those described in PCT / US93 / 04145 and US Pat. Pins based on methods such as those described in 5,288,514.

Additional flow channels or spotting methods that can attach sensor polynucleotides to a substrate are described in US patent application Ser. No. 07 / 980,523 filed November 20, 1992 and US Patent No. 5,384,261. The reagent is conveyed to the substrate by (1) fluidization in a channel defined in a predefined zone, or (2) "spotting" on a predefined zone. Protective coatings, such as hydrophilic or hydrophobic coatings (depending on the nature of the solvent) may sometimes be used on the portion of the substrate to be protected, in combination with a substance that promotes wetting by reactant solutions in other zones. In this method, the fluidizing solution further prevents passing out of their indicated flow path.

Conventional dispensers include optionally controlled robotically controlled micropipettes, ink-jet printers, series of tubes, manifolds, pipette arrays, and the like, with various reagents in sequence or simultaneously to the reaction zone. Can serve.

Excitation and Detection of Chromophores

Any instrument that can excite a polycation chromophore and provide a wavelength shorter than the emission wavelength (s) to be detected can be used for the excitation. The excitation light source preferably does not directly excite the signal chromophore directly. The light source is: broadband UV light source such as a suitable filter and deuterium lamp, output of a white light source such as xenon lamp or deuterium lamp after passing monochromator to extract desired wavelength, continuous wavelength (cw) gas laser, solid state diode laser, or any pulsed laser. The light emitted from the signal chromophore can be detected by any suitable device or technique; Several suitable approaches are known in the art. For example, a fluorometer or spectrophotometer can be used to detect whether the test sample emits light of the wavelength characteristic of the signal chromophore by excitation of the chromophore.

Kit

Kits are also provided comprising reagents suitable for carrying out the methods of the invention. In one embodiment, the kit comprises a single-stranded sensor polynucleotide complementary to the desired target polynucleotide and the polycation polyorganism. Sensor polynucleotides are conjugated to signal chromophores. If a target polynucleotide is present in the sample, the sensor polynucleotide hybridizes to the target, and consequently an increase in energy release from the signaling chromophore can be detected.

The components of the kit are held in one housing. Instructions for using the kit to perform the method of the present invention may be provided with the housing and may be provided in any fixed medium. The instructions may be located within or outside the housing and may be printed on the interior or exterior of any surface forming the housing to provide for easy reading. The kit may be in the form of a multiplex containing a plurality of one or more different sensor polynucleotides that can hybridize to corresponding different target polynucleotides.

The following examples provide a complete description of how to use and prepare the invention to those of ordinary skill in the art and do not limit the scope of the invention. Efforts have been made to ensure that the numerical values used (eg amounts, temperature, etc.) are accurate, but there should be some experimental error and deviation. On the other hand, although not indicated, parts are parts by weight, temperature is in degrees Celsius, pressure is at or near atmospheric, and all materials are commercially available.

Example 1. Identification of FRET Scheme.

Hybridization tests using energy transfer from a light harvesting chromophore system to a signaling chromophore, together with an iodide counterion, cationic water soluble conjugated polymer poly (9,9-bis (6'-) N, N, N-trimethylammonium) -hexyl) -fluorene phenylene) to demonstrate using (1). The sensor polynucleotide sequence is 5'-GTAAATGGTGTTAGGGTTGC-3 ', which has a fluorescein at position 5' and corresponds to pX02, the spore encapsulation plasmid (anthrax, Bacillus anthracis ), and oligo- C ^* form the sample. Absorption and emission spectra of ²⁴ polymers and signal chromophores are shown in FIG. The results show a photo window for the excitation of the polymer 1 between the adsorption of DNA and fluorescein. Direct excitation of the polymer (1) results in energy transfer (ET) to fluorescein as shown in FIG. The release of polymer (1) and the absorbance overlap of fluorescein were chosen to ensure FRET. The amount of ²⁵ ET can be estimated by the ratio of integrated acceptor to donor emission.

Example 2. FRET Description in the Presence of Target Polynucleotides

Hybridization of oligo-C ^* probes leads to changes in the ET ratio. The sensor polynucleotide ([oligo-C ^* ] = 2.1 × 10 ⁻⁸ M) is SEQ ID NO: 5 'in the presence of the same molar amount of 40 base pair strands comprising the complementary 20 base pair sequence 5'-CATCTGTAAATCCAAGAGTAGCAACCCTAACACCATTTAC-3' The non-complementary 40 base strands with -AAAATATTGTGTATCAAAATGTAAATGGTGTTAGGGTTGC-3 'were annealed at their T _m (58.4 ° C) up to 2 ° C in the same form. Direct comparison of the resulting fluorescens revealed an ET ratio greater than six times greater for hybridized DNA. See FIG. 2. It is also most important that the optical difference is observed in the presence of 10 mmol sodium citrate and 100 mmol sodium chloride buffer. Buffer ions weaken the electrostatic interaction between the fluorescence quencher of the opposite charge and the CPs, but block charges such as charges on complementary DNA strands that promote hybridization. The ²⁶ standard PMT is using a fluorescent spectrophotometer equipped with a xenon lamp (fluorometer) is a hybrid DNA ratio (非) - a hybrid DNA in the look Sensor polynucleotide] = 2.8 × 10 ^-9 M at least three times the rate at ET to provide.

Example 3. Optimization of Energy Transfer

Energy transfer can be optimized by varying the ratio of polymer (1) to oligo-C ^* . At the concentration [oligo-C ^* ] = 2.1 × 10 ⁻⁸ M, the initial addition of the polymer immediately raises the ET ratio and decreases as the amount of polymer (1) begins to significantly exceed the amount of oligo-C ^* . The maximum value of the ET ratio corresponds to a polymer chain to oligo-C ^* ratio almost 1: 1. Not all chains are tightly complexed to oligo-C ^* at high polymer concentrations, and donor release rises faster than that of the signaling chromophores. In the case of [polymer (1)] / [oligo-C ^* ] <1, this correlation is expected because not all sensor polynucleotide strands can effectively complex to independent polymer chains. Conversely, in the situation where [polymer (1)] / [oligo-C ^* ]> 1, all photons utilized by polymer (1) (juvener) can be transferred to oligo-C ^* (dump) It is not. Once the repeat units of polymer to oligo-C ^* reached approximately 100, the photoluminescence of the chromophore recorded no longer increased, indicating saturation of the base. At this ratio the integrated fluorescein emission is about four times greater than that of the directly excited (480 nm) probe without polymer (1), which is further evidence that the signal has been amplified.

Example 4 FRET Delivery from Multi-Generation to Polynucleotide Dye Through Sensor Polynucleotides in Improved Selectivity

Hybridization of oligo-C ^* probes (5'-Fl-ATCTTGACTATGTGGGTGCT-3 ') leads to differences in ET ratios for two different sensor chromophores. The sensor polynucleotide ([oligo-C ^* ] = 1 × ^10-8 M) is sequenced in the presence of the same molar amount of 20 base pair strands comprising the complementary 20 base pair sequences of (5′-AGCACCCACATAGTCAAGAT-3 ′). The non-complementary 20 base pair strands with (5′-CGTATCACTGGACTGATTGG-3 ′) were annealed at 2 ° C. below their T _m (58.5 ° C.) in the same form. The two DNA mixtures contain ethidium bromide ([EB] = 1.1 ×) at room temperature in potassium phosphate monohydric-sodium hydroxide buffer solution (50 mM, pH = 7.40), inserting EB within the double structure of the hybridized DNA pair. 10 ^-6 M). The addition of polymer (1) ([1] = 1.6 × 10 ⁻⁷ M) to water and subsequent excitation (380 nm) of (1) results in energy transfer to the sensor polynucleotide in (1), followed by Energy transfer takes place from the polynucleotide to the EB. Comparison of the resulting fluorescence revealed that only the emission from insertion of the dye EB appeared in the presence of the DNA to which the sensor solution was hybridized. See FIG. 3. The results indicate that the DNA sequence sensor of the present invention detects release from a polynucleotide-specific dye upon excitation of a polycation multistage group, thereby the presence of a target single-stranded DNA having a specific base sequence complementary to the sequence of the sensor polynucleotide. It will be described that can be detected. The chromophore used in this example was chosen to adequately overlap the energy between the chromophore and the two signal displaying chromophores.

Example 5. Description of Optical Amplification of Polynucleotide Dye Using FRET from a Chromophore System.

(1), in a solution of sensor polynucleotide and polynucleotide specific dye (ethidium bromide, EB), excitation of (1) is directly included in the same double-stranded DNA sequence lacking a signaling chromophore on the sensor polynucleotide ( 500 nm) results in an emission intensity of EB about 8 times greater than that of EB. Direct excitation of the signaling oligo-C ^* (480 nm) in the absence of polymer 1 provides only about four times the sensitization of the inserted EB. This example also shows evidence that the signal is amplified by FRET from a polycation polyorganism (conjugated with polymer (1)) to a diagnostic EB record. See FIG. 4.

Example 6. FRET Delivery to Polynucleotide Dye

Experiments using (1) and ethidium bromide (“EB”) as signal chromophores demonstrate that direct energy transfer from (1) to EB can occur in the presence of double stranded DNA. A sensor polynucleotide (5'-ATCTTGACTATGTGGGTGCT-3 ') lacking a signaling chromophore ([oligo] = 1 × 10 ^-8 M) is identical to a complementary 20 base pair sequence that is (5'-AGCACCCACATAGTCAAGAT-3'). In the presence of a molar amount of 20 base pair strands, it was annealed at 2 ° C. below its T _m (58.5 ° C.) in the same form as the non-complementary 20 base pair strands of sequence (5′-CGTATCACTGGACTGATTGG-3 ′). The two DNA mixtures contain ethidium bromide ([EB] = 1.1 ×) at room temperature in potassium phosphate monohydric-sodium hydroxide buffer solution (50 mM, pH = 7.40) inserting EB into the duplex structure of the hybridized DNA pair. 10 ^-6 M). Addition of polymer (1) ([1] = 1.6 × 10 ⁻⁷ M) to water followed by excitation (380 nm) of (1) to EB inserted in (1) only in the case of hybridized or double stranded DNA Energy transfer takes place. Release from the EB is only detected upon excitation of the polymer 1 in the hybridized sequence. See FIG. 5.

Although the present invention has been described in more detail with reference to preferred embodiments, any change and modification may be made to a person skilled in the art without departing from the scope or spirit of the invention with the aid of the techniques herein. It will be clear. Accordingly, the invention is limited only by the claims.

Claims (55)

Preparing a sample suspected of containing a target polynucleotide; Preparing a polycationic multichromophore capable of transferring energy to a signaling chromophore by excitation; Preparing an anionic sensor polynucleotide that is single stranded and complementary to the target polynucleotide, wherein the sensor polynucleotide is conjugated to a signaling chromophore; Contacting the sensor polynucleotide with a chromophore; By excitation of the chromophore in the absence of the target polynucleotide, the emitted light can be generated from the signaling chromophore; A large amount of emitted light is generated from the signaling chromophore by excitation of the chromophore in the presence of the target polynucleotide; Contacting the sample with the sensor polynucleotide and chromophore in solution under conditions such that the sensor polynucleotide can hybridize to the target polynucleotide (if present); Applying a light source to a solution capable of exciting the chromophore; And Detecting whether light emitted from the chromophore is increased in the presence of a sample. The method of claim 1, The chromophore is characterized in that it comprises a structure selected from the group consisting of saturated polymers, conjugated polymers, aggregates of conjugated molecules, dendrimers, and semiconductor nanocrystals. Analysis method. The method of claim 2, The chromophore comprises an saturated polymer. The method of claim 2, The chromophore comprises an dendrimer. The method of claim 2, The chromophore comprises analytical nanocrystals. The method of claim 2, The chromophore comprises an conjugated molecule. The method of claim 6, Conjugated polymer structure is represented by the following formula (1). (Formula 1) The method of claim 2, The chromophore is an aggregate of conjugated molecules. The method of claim 8, The aggregate is an analysis method characterized in that it comprises a molecule represented by the formula (2). (Formula 2) The method of claim 1, The sample is contacted with the sensor polynucleotide and the chromophore in the presence of a sufficient amount of organic solvent to reduce the hydrophobic interaction between the sensor polynucleotide and the chromophore. The method of claim 1, The sample is contacted with a number of different sensor polynucleotides having corresponding other sequences (the other sensor polynucleotides include corresponding other signaling chromophores), each of the other sensor polynucleotides corresponding to a corresponding other target polynucleotide. And optionally hybridized to. The method of claim 1, The signal displaying chromophore is an analysis method characterized in that the fluorophore (fluorophore). The method of claim 12, The fluorophore is selected from the group consisting of semiconductor nanocrystals, fluorescent dyes and lanthanide chelates. The method of claim 13, The fluorophore is an analytical method, characterized in that the semiconductor nanocrystals. The method of claim 13, Fluorescent chromophores are fluorescent dyes. The method of claim 15, Fluorophore dye is an analytical method, characterized in that fluorescein (fluorescein). The method of claim 13, The fluorophore is lanthanide chelate. The method of claim 1, The target polynucleotide is DNA. The method of claim 1, The target polynucleotide is an analysis method, characterized in that the RNA. The method of claim 1, And the sample comprises a single strand of target polynucleotide. The method of claim 1, The sample comprises a double stranded target polynucleotide. The method of claim 1, The target polynucleotide is prepared by an amplification reaction. Signal chromophore; And A polynucleotide sensing solution comprising a polycation chromophore capable of transferring energy to a signal chromophore by approaching the chromophore as an excitation, A polynucleotide sensing solution, wherein a large amount of energy can be generated from a signaling chromophore in the presence of a polynucleotide that is sensed when the chromophore is excited. Sensor polynucleotides that are single stranded and complementary to a target polynucleotide; Signal chromophore; A polycation chromophore which is capable of transferring energy to the signal chromophore by excitation when approaching the chromophore; And A sample analysis kit for a target polynucleotide comprising a housing for holding reagents of the kit, the kit comprising: The sensor polynucleotide interacts with the chromophore and, in the presence of the target polynucleotide, a sample analysis kit for the target polynucleotide, wherein a large amount of emitted light is produced by the excitation of the chromophore to be detectable from the signal chromophore. . The method of claim 1, Light emitted from a signaling chromophore above a threshold level indicates that the target polynucleotide is present in the sample. The method of claim 1, The amount of light emitted from the signaling chromophore is quantified and used to determine the amount of target polynucleotide in the sample. The method of claim 12, The fluorophore is an analysis method, characterized in that the green fluorescent protein (green fluorescent protein). The method of claim 1, The target polynucleotide is not amplified. The method of claim 1, Said method is carried out on a substrate. The method of claim 29, The substrate is selected from the group consisting of microspheres, chips, slides, multiwell plates, optical fibers, any porous gel matrix, photodiodes and optoelectronic devices. Analysis method. The method of claim 30, The substrate is a slide. The method of claim 30, The substrate is an optoelectronic device. The method of claim 30, The substrate is any porous gel matrix. The method of claim 33, wherein Analytical method characterized in that the substrate is a sol-gel (sol-gel). The method of claim 30, An analytical method wherein the substrate is nanoaddressable. The method of claim 30, The substrate is microaddressable. The method of claim 30, The substrate is conjugated to a plurality of different sensor polynucleotides having corresponding other sequences, wherein each of the other sensor polynucleotides can be selectively hybridized to the corresponding other target polynucleotide. The method of claim 1, The method comprises performing fluorescence in situ hybridization (FISH). The method of claim 22, The amplification reaction is characterized in that it comprises a polymerase chain reaction (polymerase chain reaction). The method of claim 1, Contacting a second signaling chromophore, polychromophore, and sensor polynucleotide with a sample in solution, capable of absorbing energy from the signaling chromophore in the presence of the target and emitting light; Detecting whether light emitted from the signal chromophore is increased in the presence of the sample and detecting whether light emitted from the second signal chromophore is increased in the presence of the sample. The method of claim 40, The second signaling chromophore is a polynucleotide-specific dye. A signal displaying complex formed by the method of claim 1. The method of claim 42, And a second signal chromophore. The method of claim 23, And a second signaling chromophor capable of absorbing energy from the signaling chromophore in the presence of the target and emitting light. The method of claim 44, The second signal chromophore is a polynucleotide-sensing solution, characterized in that the polynucleotide-specific dye. A substrate-binding complex formed by the analytical method of claim 29. The method of claim 46, And a second signaling chromophore. The method of claim 24, And a second signaling chromophor capable of absorbing energy from the signaling chromophore in the presence of the target and emitting light. 49. The method of claim 48 wherein And the second signaling chromophore is a polynucleotide-specific dye. The method of claim 24, And a kit having said housing that describes how to use the components of the kit for analyzing a sample for a target polynucleotide. A sensor strand that is single stranded and complementary to a target polynucleotide, wherein the sensor polynucleotide is attached to a signaling chromophore; And A polycation chromophore that is capable of transferring energy to the signal chromophore by proximity to the chromophore (the sensor polynucleotide interacts with the chromophore; By excitation of the chromophore in the absence of the target polynucleotide, the emitted light can be generated from the signaling chromophore; And a large amount of emitted light is generated from the signaling chromophore by excitation of the chromophore in the presence of the target polynucleotide. The method of claim 23, A single stranded polynucleotide sensing solution, characterized in that it further comprises a sensor polynucleotide complementary to the target polynucleotide. The method of claim 23, The signal chromophore is a polynucleotide sensing solution, characterized in that it is a polynucleotide-specific dye capable of absorbing energy from the chromophore in the presence of a target and emitting light. The method of claim 23, The signaling chromophore is a polynucleotide-specific dye that can absorb energy from the chromophore in the presence of a target and can transfer energy to a second signaling chromophore conjugated to a sensor polynucleotide that emits light. Polynucleotide sensing solution. The method of claim 3, wherein Saturated polymers are luminescent dyes.